Automated centrifugal microfluidic system for the preparation of adaptor-ligated sequencing libraries.

The intensive workload associated with the preparation of high-quality DNA libraries remains a key obstacle toward widespread deployment of sequencing technologies in remote and resource-limited areas. We describe the development of single-use microfluidic devices driven by an advanced pneumatic centrifugal microfluidic platform, the PowerBlade, to automate the preparation of Illumina-compatible libraries based on adaptor ligation methodology. The developed on-chip workflow includes enzymatic DNA fragmentation coupled to end-repair, adaptor ligation, first DNA cleanup, PCR amplification, and second DNA cleanup. This complex workflow was successfully integrated into simple thermoplastic microfluidic devices that are amenable to mass production with injection molding. The system was validated by preparing, on chip, libraries from a mixture of genomic DNA extracted from three common foodborne pathogens (Listeria monocytogenes, Escherichia coli and Salmonella enterica serovar Typhimurium) and comparing them with libraries made via a manual procedure. The two types of libraries were found to exhibit similar quality control metrics (including genome coverage, assembly, and relative abundances) and led to nearly uniform coverage independent of GC content. This microfluidic technology offers a time-saving and cost-effective alternative to manual procedures and robotic-based automation, making it suitable for deployment in remote environments where technical expertise and resources might be scarce. Specifically, it facilitates field practices that involve mid- to low-throughput sequencing, such as tasks related to foodborne pathogen detection, characterization, and microbial profiling.

Automated sample-to-answer centrifugal microfluidic system for rapid molecular diagnostics of SARS-CoV-2.

Testing for SARS-CoV-2 is one of the most important assets in COVID-19 management and mitigation. At the onset of the pandemic, SARS-CoV-2 testing was uniquely performed in central laboratories using RT-qPCR. RT-qPCR relies on trained personnel operating complex instrumentation, while time-to-result can be lengthy (e.g., 24 to 72 h). Now, two years into the pandemic, with the surge in cases driven by the highly transmissible Omicron variant, COVID-19 testing capabilities have been stretched to their limit worldwide. Rapid antigen tests are playing an increasingly important role in quelling outbreaks by expanding testing capacity outside the realm of clinical laboratories. These tests can be deployed in settings where repeat and rapid testing is essential, but they often come at the expense of limited accuracy and sensitivity. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) provides a number of advantages to SARS-CoV-2 testing in standard laboratories and at the point-of-need. In contrast to RT-qPCR, RT-LAMP is performed at a constant temperature, which circumvents the need for thermal cycling and translates into a shorter analysis time (e.g., <1 h). In addition, RT-LAMP is compatible with colorimetric detection, facilitating visualization and read-out. However, even with these benefits, RT-LAMP is not yet clinically deployed at its full capacity. Lack of automation and integration of sample preparation, such as RNA extraction, limits the sensitivity and specificity of the method. Furthermore, the need for cold storage of reagents complicates its use at the point of need. The developments presented in this work address these limitations: We describe a fully automated SARS-CoV-2 detection method using RT-LAMP, which also includes up-front lysis and extraction of viral RNA, performed on a centrifugal platform with active pneumatic pumping, a disposable, all-polymer-based microfluidic cartridge and lyophilized reagents. We demonstrate that the limit of detection of the RT-LAMP assay itself is 0.2 copies per µL using N and E genes as target sequences. When combined with integrated RNA extraction, the assay sensitivity is 0.5 copies per µL, which is highly competitive to RT-qPCR. We tested the automated assay using 12 clinical swab specimens from patients and were able to distinguish positive and negative samples for SARS-CoV-2 within 60 min, thereby obtaining 100% agreement with RT-qPCR results.

Priming and testing silicon patch-clamp neurochips.

We report on the systematic and automated priming and testing of silicon planar patch-clamp chips after their assembly in Plexiglas packages and sterilization in an air plasma reactor. We find that almost 90% of the chips are successfully primed by our automated setup, and have a shunt capacitance of between 10 pF and 30 pF. Blocked chips are mostly due to glue invasion in the well, and variability in the manual assembly process is responsible for the distribution in shunt capacitance value. Priming and testing time with our automated setup is less than 5 min per chip, which is compatible with the production of large series for use in electrophysiology experiments.